ABSTRACT:Mucoadhesive Microspheres of Bovine Insulin containing a muchoadhesive polymers Sodium Alginate and Carbapol-934 were prepared by Ionic Gelation technique and Bovine Insulin encapsulated by lyophilization by utilizing 32 Factorial Drug Design and 9 formulation prepared. The formulation C-3 was selected as a final optimized formulation for its significant characteristics. The Optimized Mucoadhesive microspheres C-3 was loaded with bioenhancers such as Sodium deoxycholate, Sodium Taurocholate and Lecithin from soya in 1% w/w. Drug release study was carried out. The mucoadhesive microspheres loaded with 1% w/w Sodium deoxycholate indicate significant release profile. Hence the final optimized formulation loaded with1% w/w Sodium deoxycholate was investigate for animal study by selecting rabbit model. The diabetes was induced by using Alloxan 150 mg/kg to rabbits. The final optimized formulation loaded with and without 1% w/w Sodium deoxycholate. The formulation without bioenhancer was remarked as reference formulation; both formulations were given to animal by nasal route, with a dose 1.5 U/Kg by selecting Crossover drug design study. Prior to investigate a optimized and reference formulation to animal a standard dose of bovine insulin was given to each animal 0.5 U/Kg by subcutaneously. Serum blood concentration of bovine insulin was estimated by using previously calibrated and validated RP-HPLC method at predetermined time schedule and pharmacokinetic study was done. The % blood glucose levels were recorded during animal study.